The present invention relates to a material having selective affinity for compounds produced by prokaryotic micro-organisms, namely gram-negative bacteria and gram-positive bacteria. From among the compounds produced by bacteria, it relates to a material which removes or results in a loss of toxic activity (detoxification) of lipoteichoic acid (hereinafter abbreviated to LTA), protein A (hereinafter abbreviated to PrA), xcex1-hemolysin (abbreviated to aHL) or proteinase. In particular, since it binds to the LTA and/or PrA and/or aHL and/or proteinase occurring in blood and other such solutions of high protein concentration, it is ideally used either as a medicinal agent which brings about a loss of the toxic activity (detoxification) of LTA and/or PrA and/or aHL and/or proteinase, or as a purification column or dressing which removes LTA and/or PrA and/or aHL and/or proteinase.
In recent years, as a result of a variety of studies, LTA has come to be regarded as one of the causal substances of gram-positive bacterial sepsis. For example, it has been reported that it induces expression of nitric oxide synthase in cultured vascular smooth muscle cells (J. Cardiovasc. Pharmacol., 20, S145-S147 (1992)), that as a result of intravenous infusion in rats there occurs a reduction in blood pressure and a reduction in the pressor reaction due to noradrenaline (Br. J. Pharmacol, 144, 1317-1323 (1995)), that as a result of intrathoracic administration to animals, neutrophil infiltration into the thoracic cavity is observed and pleurisy occurs (JP-A-9-163896) and, furthermore, that if LTA and peptidoglycan are jointly administered to rats, shock and internal organ disturbances occur (J. Exp Med., 188 (2), 305-315 (1998)). By way of contrast, it has been reported that nitric oxide production is inhibited by anti-mouse CD14 antibodies (Biochem Biophys Res Commun, 233 (2), 375-379 (1997)) or by N(omega)-nitro-L-arginine methyl ester (Infect Immun, 65 (6), 2074-2079 (1997)), and that platelet activating factor antagonist inhibits shock deaths resulting from LTA administration to mice (JP-A-9-208493). Furthermore, the effects of aminoguanidine and dexamethasone (Br J Pharmacol. 119 (7), 1411-1421 (1996)) have also been investigated. However, these all target substances produced in the latter part of the inflammatory response initiated by LTA and there are no reports of drugs which target the LTA itself. Now, regarding materials for medical use, there is a report (ASAIO J, 44 (1), 48-53 (1998) that fibre-immobilized polymyxin-B brings about a 20% reduction in the TNF-xcex1 production due to a Staphylococcus aureus culture supernatant (diluted with 10% human plasma-containing medium). However, various toxins are contained in the culture supernatant and LTA removal has not been confirmed. Moreover, the percentage removal was also low, at 20%. Thus, there have not been known hitherto materials which have a high affinity for LTA in high protein solutions such as plasma.
In addition, it is known that, as a result of the agglomeration brought about by PrA binding to immunoglobulin G (hereinafter abbreviated to IgG), which is the main protein of the human immune system, deactivation of the activity thereof is brought about. As a result of the IgG deactivation and a lowering of the immune capability, bacteria readily invade the body and sepsis is aggravated. In the same way, aHL is a protein which harms cells by bringing about the formation of pores in the cell membranes of human cells (particularly blood corpuscles) and, by destroying cells, there is created a situation where infection readily occurs and sepsis is aggravated. No materials are known which have a characteristic affinity for such toxins, PrA and aHL.
The present invention relates to a material which resolves these prior-art problems and also has further novel functions, and its objective is to provide a material which can rapidly bring about a loss of toxic activity (detoxification) of, or can remove, the LTA and/or PrA and/or aHL and/or proteinase in blood or other such solutions of high protein concentration.
Specifically, the material of the present invention has a high affinity for bacterially-derived components and binds LTA and/or PrA and/or aHL and/or proteinase present in blood, plasma and other such body fluids, or in pharmaceutical preparations and, it this way, it is possible to detoxify the activities of these toxins and to treat and prevent sepsis and infectious diseases. Moreover, where this material is water-insoluble, there can then be provided a material which adsorbs LTA and/or PrA and/or aHL and/or proteinase present in blood, plasma and other such body fluids, or in drugs, by binding such toxins, and by employing such a material there can be provided a blood purification column or a wound dressing for the treatment or prevention of sepsis or infectious disease.
The present invention has the following constitution for resolving the problems described above.
(1) A bacterially-derived component detoxification or removal material which is characterized in that it has at least one functional group capable of hydrogen bond formation and detoxifies or removes at least one of the bacterially-derived components selected from lipoteichoic acid, protein A, xcex1 hemolysin proteinase and endotoxin.
(2) A bacterially-derived component detoxification or removal material which is characterized in that it has a functional group capable of hydrogen bond formation and a hydrophobic group and/or ether bond, and it detoxifies or removes at least one of the bacterially-derived components selected from lipoteichoic acid, protein A, xcex1 hemolysin, proteinase and endotoxin.
(3) A material for sepsis treatment where a material according to (1) and (2) is employed.
(4) A wound dressing employing a material according to (1) and (2).
(5) A method for the removal or detoxification of lipoteichoic acid and/or protein A and/or a hemolysin and/or proteinase in liquids, using a material according to (1) and (2).
In the present invention, LTA refers to a substance from which the cell membrane and cell wall of gram-positive bacteria such as bacteria of the genera Streptococcus, Micrococcus, Lactobacillus, Staphylococcus, Bacillus and Enterococcus are composed (xe2x80x9cIka Saikingaku [Medical Bacteriology]xe2x80x9d, Ed. by Masanosuke Yoshikawa, Published by Nankodo). Furthermore, PrA and aHL are proteins produced by Staphylococcus aureus. Again, in the present invention, proteinase refers to bacterially-derived proteinase which cleaves partial sequences from protein precursors, producing active proteins. These bacterially-derived components are toxins which are highly likely to be involved in the aggravation of infectious diseases, in particular sepsis.
In addition, it is suspected that there is a relationship between streptolysin, coagulase, enterohemorrhagic toxin, pseudomonas exotoxin A, cholera toxin, botulinus toxin, verotoxin, leukocidin, superantigen, endotoxin and the like, and the aggravation of infectious diseases, in particular, sepsis. Of these, superantigen and endotoxin are highly toxic and the detoxification or removal of these toxins is desirable at the same time as that of LTA, PrA and aHL.
In the present invention, there are no particular restrictions on the functional groups capable of hydrogen bond formation, and examples are the urea bond, thiourea bond, urethane bond, amide group, amino group, hydroxyl group, carboxyl group, aldehyde group, mercapto group and guanidino group, but possession of at least one urea bond, thiourea bond, amide bond, amino group or hydroxyl group is preferred. There are no particular restrictions on the structure adjoining the group capable of forming a hydrogen bond, and there can be employed aliphatic compounds such as propane, hexane, octane and dodecane, or alicyclic compounds such as cyclohexane and cyclopentane but, taking into consideration their high affinity, aromatic compounds such as benzene, naphthalene or anthracene are more preferably used. Derivatives such as bromoheptane, chlorocyclohexane, methylbenzene, chlorobenzene, nitrobenzene, diphenylmethane and chloronaphthalene are also suitably employed. Again, it is further preferred that there be at least two groups capable of hydrogen bond formation and, in particular, there is preferably employed a structure which also possesses a functional group capable of forming a hydrogen bond, such as an amino group, hydroxyl group or carboxyl group, as the structure adjoining the urea bond, thiourea bond or amide bond. As examples of compounds with an amino group, there are aminohexane, monomethylaminohexane, dimethylaminohexane, aminooctane, aminododecane, amino-diphenylmethane, 1-(3-aminopropyl)imidazole, 3-amino-1-propene, aminopyridine, aminobenzenesulphonic acid, tris(2-aminoethyl)amine and the like, more preferably diaminoethane, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, dipropylenetriamine, polyethyleneimine, Nxe2x80x2-methyl-2,2xe2x80x2-diaminodiethylamine, N-acetylethylenediamine, 1,2-bis(2-aminoethoxyethane) and other such compounds with a plurality of amino groups (sometimes referred to as polyamines). Again, as examples of compounds with a hydroxyl group, there can be used hydroxypropane, 2-ethanolamine, 1,3-diamino-2-hydroxypropane, hydroxybutanone, hydroxybutyric acid, hydroxypyridine and the like, glucides such as monosaccharides, oligosaccharides and polysaccharides such as lucose, glucosamine, galactosamine, maltose, cellobiose, sucrose, agarose, cellulose, chitin, chitosan and the like, and derivatives of these. Furthermore, as examples of structures with a carboxyl group, there can be used xcex2-alanine, n-caproic acid, isobutyric acid, xcex3-amino-xcex2-hydroxybutyric acid and the like. Most preferably, there can be used as the material of the present invention a compound both an aromatic group and a compound capable of forming a hydrogen bond as structures adjoining the urea, thiourea or amide groups.
As a functional group other than a group capable of forming a hydrogen bond, it is preferred that there be present a hydrophobic group or ether bond. Hydrocarbons with at least 4 carbons and aromatic rings are preferred as the hydrophobic group, and they are effective without distinction in terms of being linear, branched, cyclic, saturated or unsaturated. Specific examples are the n-butyl group, sec-butyl group, tert-butyl group, isobutyl group, n-hexyl group, n-octyl group, n-dodecyl group, cyclohexyl group, benzyl group, 3,3-diphenylpropyl group n-butylamino group, n-hexylamino group, n-dodecylamino group, n-hexadecylamino group and the like.
As the hydrophobic group, it is preferred that it have log P value at least 0.7 (P=partition coefficient in an octanol/water system). It is possible to refer to the published values of a number of known compounds (see Albert Leo, Corwin Hansh and David Elkins, Partition coefficients and their uses, Chemical Reviews, vol.71, No.6 p525-616 (1971)).
As examples of structures containing an ether bond or ether bonds, there are straight chain ethers such as the ethoxyethyl group and methoxyethyl group, cyclic ethers such as crown ethers, and the ethers contained in glucides such as cellulose, agarose and the like.
Furthermore, it is also possible to use polyureas, polythioureas and polyamides, which contain a plurality of urea bonds, thiourea bonds and amide bonds in the molecular structure, as the material of the present invention. Here too, any of the aforesaid structures can be used as structures adjoining the urea bonds, thiourea bonds or amide bonds, but it is most preferred that there be used both an aromatic compound and a group (or groups) capable of forming a hydrogen bond, such as a compound which possess hydroxyl, amino or carboxyl groups (including glucides or derivatives thereof).
Moreover, the material of the present invention may also be either monomer, oligomer or polymer, so material where an aforesaid structure or part thereof has been polymerized is also included in the materials of the present invention. Thus, as the aforesaid structure or part thereof, there can be suitably employed synthetic polymers such as nylon, polymethyl methacrylate, polysulphone, polystyrene, polyethylene, polyvinyl alcohol, polytetrafluoroethylene and the like, or natural polymers including cellulose, collagen, chitin, chitosan and derivatives of these, etc. That is to say, there is ideally carried out the introduction of groups capable of hydrogen bond formation into homopolymer, copolymer or blended such synthetic polymers or natural polymers. Furthermore, there can also be appropriately used an inorganic material such as metal, ceramic or glass which has been covered by a suitable polymer. The term carrier in the present invention denotes a support on which the material of the present invention is fixed, and it may be for example a synthetic polymer or natural polymer as exemplified above.
The material of the present invention can be synthesized by generally-known methods. For example, in the case where a urea bond or thiourea bond is introduced into an aliphatic compound or an aromatic compound, there can be used the method of performing reaction between an amino compound and an isocyanate compound or isothiocyanate compound. Furthermore, in the case of the introduction of an amide group into an aliphatic compound or aromatic compound, there can be used for example the method of performing reaction between an acid, acid chloride or acid anhydride and an amino compound. Any mixing ratio of amino compound and isocyanate compound, isothiocyanate compound, acid, acid chloride or acid anhydride can be selected but, normally, there is desirably used 0.1 to 10 mol of the amino compound per 1 mol of the isocyanate compound, isothiocyanate compound, acid, acid chloride or acid anhydride. As the isocyanate compound or isothiocyanate compound, there can be used any aliphatic isocyanate compound or isothiocyanate compound such as, for example, ethyl isocyanate, stearyl isocyanate, n-butyl isocyanate, iso-butyl isocyanate, n-propyl isocyanate, methyl isothiocyanate, ethyl isothiocyanate, n-butyl isothiocyanate, benzyl isothiocyanate, hexamethylene diisocyanate, cyclohexyl isocyanate, cyclohexyl isothiocyanate, cyclohexyl isothiocyanate, cyclohexyl diisocyanate and the like, but more preferably there is used an aromatic isocyanate compound or isothiocyanate compound such as phenyl isocyanate, chlorophenyl isocyanate, fluorophenyl isocyanate, bromophenyl isocyanate, nitrophenyl isocyanate, tolyl isocyanate, methoxyphenyl isocyanate, 1-naphthyl isocyanate, 4,4xe2x80x2-diphenylmethane diisocyanate, 3,3,5,5xe2x80x2-tetraethyl-4,4xe2x80x2-diisocyanatodiphenylmethane, phenyl isothiocyanate, chlorophenyl isothiocyanate, fluorophenyl isothiocyanate, nitrophenyl isothiocyanate, tolyl isothiocyanate, methoxyphenyl isothiocyanate, 1-naphthyl isothiocyanate and the like. As the acid chloride, there can be used any aliphatic acid chloride such as, for example, isovaleryl chloride, stearoyl chloride, cyclohexanecarbonylchloride, 6-chloronicotinyl chloride and the like but, more preferably, there can be used aromatic acid chlorides such as benzoyl chloride, 3,4-dichlorobenzoyl chloride, nitrobenzoyl chloride, 4-chlorobenzoyl chloride, 4-toluoyl chloride, benzo-[b]thiophene-2-carbonyl chloride and the like. Again, as the acid anhydride, there can desirably be used acetic anhydride, succinic anhydride, phthalic anhydride, benzoic anhydride and the like. Furthermore, the amino group in the amino compounds used in the present invention can be a primary amino group, secondary amino group or tertiary amino group and, as the amino compound, there can desirably be used, for example, ammonia, sec-octylamine, 1-(3-aminopropyl)imidazole, 3-amino-1-propene, aminopyridine, aminobenzenesulphonic acid, tris(2-aminoethyl)amine and the like. Again, there can also be used advantageously a polyamino compound or an amino compound which possesses a hydroxyl group or carboxyl group, such that it is possible to introduce a group or groups capable of forming a hydrogen bond in addition to the urea, thiourea or amide bonds. As the polyamino compound, there can be used any of, for example, diaminoethane, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, dipropylenetriamine, Nxe2x80x2-methyl-2,2xe2x80x2-diaminodiethylamine, polyethyleneimine, N-acetyl-ethylenediamine, 1,2-bis(2-aminoethoxy)ethane and the like. As an amino compound with a hydroxy group, there can be used 2-ethanolamine, 3-propanolamine, 6-hexanolamine, 1,3-diamino-2-hydroxypropane, 2-(2-aminoethoxy)ethanol, 2-(2-aminoethylamino)ethanol, glucamine, N-methyl-1,3-diaminopropanol or other such aliphatic amine, or 4-aminophenol, diaminophenol, aminohydroxypyrimidine, diaminohydroxypyrimidine, diaminohydroxypyrazole or other such aromatic amine, or serine, tyrosine or other such amino acid. Again, by the reaction of epichlorohydrin and an amino compound, or 1,3-dibromo-2-hydroxypropane with a compound having only an amino group it is possible to synthesize an amino compound with a hydroxyl group from a compound having only a hydroxyl group, or a compound only having an amino group. Furthermore, it is possible to use the same methods as above in the case where a group capable of forming a hydrogen bond is introduced into a glucide. That is to say, in the case of a glucide with an amino group or amino groups such as chitosan or glucosamine, reaction can be carried out as described above with isocyanate compounds, isothiocyanate compounds, acids, acid chlorides or acid anhydrides. In the case of a glucide which does not possess amino groups, amino groups can be introduced by activating hydroxyl groups in the glucide with epichlorohydrin or tresyl chloride, and then reacting with ammonia, diaminoethane or the like. By utilizing such amino groups, it is then possible to introduce groups capable of forming hydrogen bonds such as urea bonds, thiourea bonds, amide bonds or the like into the glucide. As amino compounds with a carboxyl group, there can be used for example xcex2-alanine, 4-amino-n-butyric acid, xcex3-amino-xcex2-hydroxy-n-butyric acid, 6-amino-n-caproic acid or the like.
Furthermore, in the case where the material of the present invention is an oligomer or polymer, there is preferably used the method of performing reaction between the amino group of a compound with a group which possesses a hydrogen bond forming capability with oligomer or polymer which has isocyanate groups, carboxyl groups or carboxylic acid active ester groups such as succinimide groups. Reacting an aforesaid isocyanate compound, isothiocyanate compound, acid, acid chloride or acid anhydride with an amino group-possessing oligomer or polymer, or oligomer or polymer in which amino groups have been introduced using ammonia, diaminoethane, 1,3-diaminopropane, 1,3-diamino-2-hydroxypropane, 1,2-bis(2-aminoethoxy)ethane, tris(2-aminoethyl)amine, 2-(2-aminoethylamino)ethanol or the like, is also a preferred method. Furthermore, controlling the reaction time, reaction temperature or mixing ratio, etc, or using protective groups, so that the acid chloride or isocyanate compound does not react with a group or groups capable of forming a hydrogen bond, other than the amino group or groups, is desirable. Where required, functional groups such as amino groups, isocyanate groups, carboxyl groups or carboxylic acid active ester groups such as succinimide groups can be introduced into the oligomer or polymer.
Moreover, in the case where the material of the present invention is a polyurea or polythiourea, it is possible to use the method of performing reaction between, for example, a polyisocyanate compound or polyisothiocyanate compound and a polyamino compound. Normally, in terms of the amount of reagents, there is desirably used from 0.1 to 10 mol of the polyamine per 1 mol of the polyisocyanate compound or polyisothiocyanate compound. As the polyisocyanate compound or polyisothiocyanate compound there is ideally employed hexamethylene-diisocyanate, cyclohexyldiisocyanate, tolylene diisocyanate, 4,4xe2x80x2-diphenylmethanediisocyanate, 3,3xe2x80x2,5,5xe2x80x2-tetraethyl-4,4xe2x80x2-diisocyanatodiphenylmethane, xylene-diisocyanate, methylenebis(4-phenylisothiocyanate) or the like. Again, as the polyamino compound, there can be desirably employed diaminoethane, diaminopropane, 1,3-diamino-2-hydroxypropane, Nxe2x80x2-methyl-1,3-diamino-2-propanol, diaminophenol, N,Nxe2x80x2-diaminopiperazine, diethylenetriamine, triethylenetetramine, tetraethylene-pentamine, polyethyleneimine, dipropylenetriamine, Nxe2x80x2-methyl-2,2xe2x80x2-diaminodiethylamine or the like. Furthermore, in the case where the material of the present invention is a polyamide, there can be used for example the method of polycondensation of a polycarboxylic acid and a polyamine. Again, in the case of polyureas, polythioureas and polyamides, there is also advantageously carried out the method where no polyisocyanate, polyisothiocyanate, polycarboxylic acid, or the like, is used, and each functional group is introduced, one at a time, in turn, to finally obtain the polyurea, polythiourea or polyamide.
Again, the introduction of the hydrophobic group or groups can be carried out by known methods. Thus, preparation can readily be performed by carrying out reaction between a material with an amino group and the halide of the hydrocarbon compound which constitutes the hydrophobic group, for example 1-bromobutane, 1-bromohexane or the like, or by firstly performing activation of the hydroxyl groups of a hydroxyl group compound using epichlorohydrin, tresyl chloride or the like, after which reaction is carried out with the amino derivative of a hydrocarbon compound, for example 1-aminobutane, 1-aminohexane or the like.
Moreover, ether bonds can be introduced by the reaction between bis(2-amino)ethoxyethane, polyoxyethylenebisamine or an amino sugar such as glucosamine and activated ester groups or haloalkyl groups in the material.
All the above reactions are carried out, as a rule, at a reaction temperature of 0 to 150xc2x0 C. and a reaction time of 0.1 to 24 hours. Furthermore, while it is not absolutely necessary to employ a reaction solvent, in general the reaction is carried out in the presence of solvent. As examples of the solvent, there are methanol, ethanol, isopropyl alcohol, n-butanol, hexane, acetone, N,N-dimethylformamide, dimethylsulphoxide and other such aliphatic hydrocarbons, benzene, toluene, xylene and other such aromatic hydrocarbons, dichloromethane, chloroform, chlorobenzene and other such halogenated hydrocarbons, diethyl ether, tetrahydrofuran, dioxane and other such ethers. Where required, following the end of the reaction, the reaction liquid is subjected to the usual post-treatment such as filtering and concentration, after which it can be purified by column chromatography, recrystallization or other such process. Furthermore, in the case of a water-insoluble material, washing using a glass filter or the like is also a preferred method.
Amongst the materials of the present invention, those that are water insoluble are favourably employed as, for example, a dressing or a column for removing LTA and/or PrA and/or aHL and/or proteinase. The form thereof is not particularly restricted but, in the case where used as a column, a form such as beads, fibre, hollow fibre, fibre bundles, yarn, net, knitted material, woven fabric or the like is preferred. In the case of a dressing, a woven material or film form is preferred. Furthermore, the material need not just be used on its own, but it can also be used as one column or dressing, immobilized on a suitable material or mixed with some other material. The immobilizing or mixing stage, etc, may be carried out prior to, or after, processing in the aforesaid form. In the case where a column employing the material of the present invention is used as a column for extracorporeal circulation, the blood led out from the body may be directly passed through the column or this may be used in combination with a plasma separation membrane or the like.